Chemiluminescent protein chip, kit and detection method for detection of fucose index of seroglycoid

A protein chip and chemiluminescence technology, applied in the field of protein detection, can solve the problems of increasing the complexity of operations, high technical requirements of the i30 detection system, and limited popularization and application, so as to reduce the detection cost and expense, reduce the amount of blood samples and antibodies, The effect of improving detection efficiency

Inactive Publication Date: 2015-08-19
BEIJING YOUAN HOSPITAL CAPITAL MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, plant lectin affinity immunoelectrophoresis technology and The i30 detection system has high technical requirements, cumbersome operation, and expensive reagents, which limit its popularization and application
However, the glycosyl capture spin column increases the complexity of the operation because the sample processing and detection are carried out separately.

Method used

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  • Chemiluminescent protein chip, kit and detection method for detection of fucose index of seroglycoid
  • Chemiluminescent protein chip, kit and detection method for detection of fucose index of seroglycoid
  • Chemiluminescent protein chip, kit and detection method for detection of fucose index of seroglycoid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1. Protein chip preparation and use process

[0059] Reagents and instruments used in the experiment: mouse monoclonal antibody AFP (Shenzhen Faipeng Company); lentilin (Sigma Company); aldehyde-based chip (Shanghai Biopro Company); biotin-labeled rabbit-derived primary antibody (Abcam Company, USA) ; Avidin HRP (US abcam company), chemiluminescent scanner. (Developed by the laboratory of Professor Wang Shengqi, Academy of Military Medical Sciences)

[0060] PBS formula: sodium chloride (NaCl) 8g, potassium chloride (KCl) 0.2g, disodium hydrogen phosphate (Na2HPO4) 1.44g, potassium dihydrogen phosphate (KH2PO4) 0.24g, adjust pH to 7.4, constant volume 1L

[0061] PBST formula: PBS, 1L+Tween-20, 1ml

[0062] The chip is an aldehyde-based chip (Shanghai BioPro Company), each chip contains 10 detection grids (detection sub-areas), each grid detects one serum, and 10 serums are tested at a time.

[0063] In each detection grid, mouse-derived monoclonal antibody A...

Embodiment 2

[0071] Embodiment 2. Establishment of the detection method of the present invention

[0072] (1) Standard curve and regression equation a.

[0073] Using the purchased AFP antigen (abcam company in the United States), set to different concentration gradients, (1-5) 80ng / ml, 40ng / ml, 20ng / ml, 10ng / ml, 5ng / ml, 6. Liver cancer serum, 7. Liver cancer serum, 8 blank controls, 9 healthy serum, 10 liver cancer serum ( image 3 , the chip antibody spotting antibody is AFP A: 1mg / ml; B: 0.5mg / ml; C: 0.25mg / ml).

[0074] The operation process and protein chip in Example 1 are used to detect each concentration gradient of AFP standard substance, and the detection scan results are shown in image 3 . The test results are drawn as a standard curve, with the concentration of the standard substance as the abscissa and the pixel value as the ordinate, and the standard curve is drawn on the coordinate paper. Find the corresponding concentration from the standard curve according to the pixe...

Embodiment 3

[0080] Embodiment 3. Stability, accuracy and reliability of sample detection test method of the present invention

[0081] Serum samples:

[0082] 39 liver cancer sera: from the specimen bank of You'an Hospital Affiliated to Capital Medical University;

[0083] 32 normal healthy human serum;

[0084] 9 blank controls (blank control is 1×PBS).

[0085] The detection process is the same as in Example 1.

[0086] Substitute the sample's pixel values ​​into Figure 4 The regression equation a calculates the AFP concentration of the sample, and then multiplies it by the dilution factor, which is the total AFP concentration of the sample. Substitute the sample's pixel values ​​into Figure 7 The regression equation b of the sample AFP-L3 concentration is calculated, and then multiplied by the dilution factor, which is the total concentration of AFP-L3 in the sample.

[0087] Each chip has 10 detection sub-areas, including healthy serum samples, liver cancer serum samples, and ...

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Abstract

The invention relates to a chemiluminescent protein chip, kit and detection method for detection of the fucose index of seroglycoid, belonging to the field of protein detection technology. The chemiluminescent protein chip is characterized in that a substrate slide of the protein chip at least comprises a detection subdomain; one detection subdomain is used for detection of one serum sample; each detection subdomain is provided with two detection spot areas and one row of contrast spot areas; one of the detection spot areas has detection spots formed by specific antibodies used for immobilizing alpha fetoprotein, and the other of the detection spot areas has detection spots formed by immobilization of Lens culinaris agglutinin; the contrast spot areas have contrast spots formed by immobilization of bovine serum albumin; and concentrations of substances on the detection spots in a same detection spot area are identical. The protein chip, kit and method provided by the invention can realize accurate and high flux detection of the fucose index of seroglycoid and have the advantages of high sensitivity, saving of time, convenience, economic performance and the like in clinical application.

Description

technical field [0001] The invention relates to protein detection technology, in particular to a chemiluminescent protein chip and a detection method for detecting serum glycoprotein fucose index. Background technique [0002] The sugar chain structure of alpha fetoprotein (AFP) produced by primary liver cancer and benign liver diseases such as hepatitis and cirrhosis is very different. The sugar index is much higher. Fucose has the property of binding to lentilin. AFP can be divided into AFP-L1, AFP-L2 and AFP-L3 according to its (fucosyl) affinity for lentil lectin. Among them, AFP-L1 mainly comes from benign liver diseases, AFP-L2 mainly comes from pregnant women, and AFP-L3 is the fucose glycosylated form of alpha-fetoprotein, which mainly comes from HCC. In 2005, FDA officially approved AFP-L3 as one of the markers of primary liver cancer. AFP-L3 has high specificity and sensitivity in early diagnosis, differential diagnosis, curative effect evaluation and prognosis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N21/76G01N33/57438G01N33/68G01N2800/50G01N2800/7028G01N2800/52G01N2800/085B01J19/0046G01N33/5308G01N2333/42B01J2219/00533B01J2219/00605B01J2219/00621B01J2219/00662B01J2219/00693B01J2219/00725G01N33/54366G01N2333/471G01N2440/38G01N33/54353G01N33/57488
Inventor 李宁张爱英王升启柯杨张永宏
Owner BEIJING YOUAN HOSPITAL CAPITAL MEDICAL UNIV
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